Bottom Line:
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

ABSTRACTPhospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.

Figure 1: Structure of the PHGPx gene, and of the three types of PHGPx mRNA. The structure of the mouse PHGPx gene (Accession No. AB030643) includes eight exons in the upper panel, while exons of the three types of PHGPx mRNA are shown in the lower panel. Exon Ia (gray box) includes the translational first start site ATG (+1) for mitochondrial PHGPx, and the second ATG (+82) for non-mitochondrial PHGPx. Exon Ib (shaded box) includes the translational third start site ATG (+418) for nucleolar PHGPx. The reverted open triangles show three transcriptional start sites by analysis of 5'RACE as previously reported [3]; position −147 is the transcriptional start site for mitochondrial PHGPx (M), position +25 for non-mitochondrial PHGPx (C) and position +406 for nucleolar PHGPx (N). TGA in exon III encodes selenocysteine and TAG in exon VII encodes the stop codon. SECIS is the selenocysteine insertion sequence.

Mentions:
Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique intracellular antioxidant enzyme that directly reduces peroxidized phospholipids that have been produced in cell membranes [2]. Three types of PHGPx, mitochondrial (M), non-mitochondrial (C) and nucleolar PHGPx (N), are transcribed from one gene by alternative transcription as shown in Fig. 1 [3, 4]. The gene for mouse PHGPx consists of 8 exons, with different first exons for the different types: exon Ia for mitochondrial PHGPx and non-mitochondrial PHGPx and exon Ib for nucleolar PHGPx. The mitochondrial targeting signal of PHGPx and the second start codon of non-mitochondrial PHGPx are in exon Ia of PHGPx genomic DNA [5]. After cleavage of the N-terminal mitochondrial import sequence of mitochondrial PHGPx, the mature mitochondrial protein becomes identical to the 20 kDa non-mitochondrial PHGPx [6]. Nucleolar PHGPx was first identified as a sperm nucleus-specific 34 kDa selenoprotein (called snGPx, for sperm nucleus-specific glutathione peroxidase) or nuclear PHGPx [7]. It is formed by use of an alternative promoter and start codon localized in exon Ib of the PHGPx gene [3, 7, 8]. We have previously shown that by using an N-terminal nucleolar import signal this 34 kDa PHGPx is localized in nucleoli in several cell lines [9]. We chose the name nucleolar PHGPx since non-mitochondrial 20 kDa PHGPx exists not only in the cytosol, but also in the nucleus [10].

Figure 1: Structure of the PHGPx gene, and of the three types of PHGPx mRNA. The structure of the mouse PHGPx gene (Accession No. AB030643) includes eight exons in the upper panel, while exons of the three types of PHGPx mRNA are shown in the lower panel. Exon Ia (gray box) includes the translational first start site ATG (+1) for mitochondrial PHGPx, and the second ATG (+82) for non-mitochondrial PHGPx. Exon Ib (shaded box) includes the translational third start site ATG (+418) for nucleolar PHGPx. The reverted open triangles show three transcriptional start sites by analysis of 5'RACE as previously reported [3]; position −147 is the transcriptional start site for mitochondrial PHGPx (M), position +25 for non-mitochondrial PHGPx (C) and position +406 for nucleolar PHGPx (N). TGA in exon III encodes selenocysteine and TAG in exon VII encodes the stop codon. SECIS is the selenocysteine insertion sequence.

Mentions:
Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique intracellular antioxidant enzyme that directly reduces peroxidized phospholipids that have been produced in cell membranes [2]. Three types of PHGPx, mitochondrial (M), non-mitochondrial (C) and nucleolar PHGPx (N), are transcribed from one gene by alternative transcription as shown in Fig. 1 [3, 4]. The gene for mouse PHGPx consists of 8 exons, with different first exons for the different types: exon Ia for mitochondrial PHGPx and non-mitochondrial PHGPx and exon Ib for nucleolar PHGPx. The mitochondrial targeting signal of PHGPx and the second start codon of non-mitochondrial PHGPx are in exon Ia of PHGPx genomic DNA [5]. After cleavage of the N-terminal mitochondrial import sequence of mitochondrial PHGPx, the mature mitochondrial protein becomes identical to the 20 kDa non-mitochondrial PHGPx [6]. Nucleolar PHGPx was first identified as a sperm nucleus-specific 34 kDa selenoprotein (called snGPx, for sperm nucleus-specific glutathione peroxidase) or nuclear PHGPx [7]. It is formed by use of an alternative promoter and start codon localized in exon Ib of the PHGPx gene [3, 7, 8]. We have previously shown that by using an N-terminal nucleolar import signal this 34 kDa PHGPx is localized in nucleoli in several cell lines [9]. We chose the name nucleolar PHGPx since non-mitochondrial 20 kDa PHGPx exists not only in the cytosol, but also in the nucleus [10].

Bottom Line:
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids.PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription.All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa.

ABSTRACTPhospholipid hydroperoxide glutathione peroxidase (PHGPx) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. PHGPx is transcribed from one gene into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar PHGPx by alternative transcription. In this review, we focus on our recent experiments on the regulation of promoter activity of the types of PHGPx and on the novel strategy of functional analysis of a PHGPx knockout mice model using the transgenic rescue method and Cre-LoxP system. PHGPx is especially high in testis and spermatozoa. A deficiency is implicated in human infertility. We established spermatocyte-specific PHGPx knockout (KO) mice using a Cre-loxP system. Targeted disruption of all exons of the PHGPx gene in mice by homologous recombination caused embryonic lethality at 7.5 days post coitum. The PHGPx-loxP transgene rescued PHGPx KO mice from embryonic lethality. These rescued floxed PHGPx mice were mated with spermatocyte specific Cre expressing mice. All the spermatocyte-specific PHGPx KO male mice were infertile and displayed a significant decrease in the number of spermatozoa and significant reductions in forward motility by mitochondrial dysfunction of spermatozoa. These results demonstrate that depletion of PHGPx in spermatozoa may be one of the causes of male infertility in mice and humans.